Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway

Abstract

The habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study leftright asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits α2 and β4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patchclamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slowrising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain.ateralization of brain function is found throughout the animal kingdom, yet knowledge of the underlying neural bases for left-right (L-R) specializations is limited. Multiple developmental mechanisms could account for differential neural activity, including asymmetry in cell number or density, in specification of neuronal types, or in connectivity. Increasing evidence suggests that L-R differences in neurotransmitter distribution also play an important role in lateralized behaviors.The bilaterally paired habenular nuclei (Hb) of zebrafish provide a valuable model to study brain asymmetry. In larvae, the dorsal habenulae (dHb) [equivalent to medial habenular nuclei (mHb) of mammals] exhibit pronounced L-R differences in size, molecular properties, and connections with their midbrain target, the interpeduncular nucleus (IPN) (1-3). The Hb-IPN pathway connects limbic areas of the forebrain and midbrain (4), and has been implicated in nicotine addiction in mammals (5).The mHb-IPN tract is a major cholinergic system across vertebrates (6-11); however, it is still unresolved whether this pathway is cholinergic in zebrafish. Cholinergic neurons are defined by the presence of choline acetyltransferase (ChAT), which synthesizes ACh from choline and acetyl CoA, and vesicular acetylcholine transporter (VAChT), which packages ACh into synaptic vesicles. The chat and vacht genes are situa...

Asymmetry of the Brain: Development and Implications

“…As shown in humans (55,66), functional lateralization of the animal brain is further suggested by reports of asymmetrical distribution of neurotransmitters in rodents (9,123) and zebrafish (42,79). Asymmetries at the structural level have also been widely reported.…”

“…Within the dorsal habenula (dHb), two main subnuclei have been defined that display LR differences in molecular markers and innervation profiles: a medial subdivision [dHbm (green nuclei in Figure 2), originally defined by the expression of the Tg(brn3a:GFP) transgene] is bigger in the right habenula (RHb), projects predominantly to the ventral region of the interpeduncular nucleus (IPN), and contains cholinergic and glutamatergic neurons; a lateral subdivision [dHbl (purple nuclei in Figure 2)] expresses the kctd12.1 [(potassium channel tetramerization domain 12.1)/left-over gene] is larger on the left side, predominantly innervates the dorsal part of the IPN, and contains mostly glutamatergic neurons (2,13,58,79). However, this classification is oversimplified, as further analyses of new habenular markers indicate that the dHb is actually made of more than these two subdomains (42).…”

Left Habenular Activity Attenuates Fear Responses in Larval Zebrafish

“…All dHb neurons are glutamatergic in zebrafish larvae, but the right nucleus contains larger populations that are also cholinergic and somatostatinergic [18, 19]. The predominance of neurons in the left dHb that are activated post-shock suggests that they are not cholinergic but rather are solely glutamatergic or express other asymmetrically distributed co-transmitters such as Substance P (SP) [19].…”

Ancestral Circuits for the Coordinated Modulation of Brain State

“…Secondary antibodies used were donkey anti-mouse Alexa647 (Jackson Immuno Research, 715-606-151), donkey anti-rabbit Alexa 594 (Jackson Immuno Research, 711-586-152), donkey anti-rat DyLight405 (Jackson Immuno Research, 712-475-153), donkey anti-rat Alexa647 (Jackson Immuno Research, 712-606-153), donkey anti-goat DyLight405 (Jackson Immuno Research, 705-475-147), and donkey anti-goat Alexa647 (Jackson Immuno Research, 705-606-147). The labeling pattern observed with these antibodies closely resembled expression patterns of genes encoding these proteins or other neurotransmitter-related proteins, as previously observed with in situ hybridization (antibody target, gene ): hypocretin- hcrt (Prober et al, 2006), cocaine- and amphetamine-regulated transcript (CART)- cart2 (Thisse and Thisse, 2004), galanin- gal (Podlasz et al, 2012), neuropeptide Y- npy (Mathieu et al, 2002), neuropeptide VF- npvf (Yelin-Bekerman et al, 2015), somatostatin- sst1.1 (Herget and Ryu, 2015), serotonin- slc6a4a/b (Norton et al, 2008), tyrosine hydroxylase- th1 (Ryu et al, 2006; Filippi et al, 2010), dopamine beta-hydroxylase- dbh (Holzschuh et al, 2003; Filippi et al, 2010), and choline acetyltransferase- vachta (Hong et al, 2013). …”